Antioxidant and food additive BHA prevents TNF cytotoxicity by acting as a direct RIPK1 inhibitor

Abstract

Butylate hydroxyanisole (BHA) is a synthetic phenol that is widely utilized as a preservative by the food and cosmetic industries. The antioxidant properties of BHA are also frequently used by scientists to claim the implication of reactive oxygen species (ROS) in various cellular processes, including cell death. We report on the surprising finding that BHA functions as a direct inhibitor of RIPK1, a major signaling hub downstream of several immune receptors. Our in silico analysis predicts binding of 3-BHA, but not 2-BHA, to RIPK1 in an inactive DLG-out/Glu-out conformation, similar to the binding of the type III inhibitor Nec-1s to RIPK1. This predicted superior inhibitory capacity of 3-BHA over 2-BHA was confirmed in cells and using in vitro kinase assays. We demonstrate that the reported protective effect of BHA against tumor necrosis factor (TNF)-induced necroptotic death does not originate from ROS scavenging but instead from direct RIPK1 enzymatic inhibition, a finding that most probably extends to other reported effects of BHA. Accordingly, we show that BHA not only protects cells against RIPK1-mediated necroptosis but also against RIPK1 kinase-dependent apoptosis. We found that BHA treatment completely inhibits basal and induced RIPK1 enzymatic activity in cells, monitored at the level of TNFR1 complex I under apoptotic conditions or in the cytosol under necroptosis. Finally, we show that oral administration of BHA protects mice from RIPK1 kinase-dependent lethality caused by TNF injection, a model of systemic inflammatory response syndrome. In conclusion, our results demonstrate that BHA can no longer be used as a strict antioxidant and that new functions of RIPK1 may emerge from previously reported effects of BHA.

p62 Promotes Survival and Hepatocarcinogenesis in Mice with Liver-Specific NEMO Ablation

“…It is also worth noting that although Nrf2ca expression did not attenuate liver damage in NEMO LPC-KO mice, feeding with the potent Nrf2 activator BHA did prevent liver damage in these mice [5,49]. Interestingly, BHA was recently reported to inhibit RIPK1 kinase activity independent of its ROS scavenging activity [66]. Considering that the liver damage in NEMO LPC-KO mice is largely RIPK1 kinase activity dependent [4], it is possible that the beneficial effect of BHA in this model is at least in part due to RIPK1 inhibition rather than Nrf2 activation.…”

Small-Molecule Receptor-Interacting Protein 1 (RIP1) Inhibitors as Therapeutic Agents for Multifaceted Diseases: Current Medicinal Chemistry Insights and Emerging Opportunities

“…BHA generally comprises an isomeric mixture of 2-BHA ( 50) and 3-BHA (51) (Figure 10A). 8 As a synthetic phenol, its antioxidant properties are commonly used in scientific research. Recently, BHA has been found to inhibit RIP1 activity, thereby protecting cells against TNF-mediated RIP1 kinase-dependent necroptosis.…”

“…Recently, the development of RIP1 inhibitors has been focused on optimizing the inhibitor kinase selection specificity, effectiveness, and pharmacokinetic (PK) properties. As RIP1 is a newly discovered and potential multifaceted-disease-related drug target, ,,,− many new RIP1 kinase inhibitors with high selectivity have been reported, and some of these inhibitors have been entered into clinical trials, such as a phase I clinical trial for ALS and phase II clinical trials for psoriasis, RA, and ulcerative colitis (UC). , Based on the current understanding of RIP1 regulatory mechanisms and recent evidence indicating pathological roles for RIP1 in human diseases, we highly anticipate that our review of the recent developments of small-molecule inhibitors targeting the RIP1 kinase and perspectives on related drug development strategies will contribute to future research into the kinase inhibitor field.…”

“…The specificity of RIP1-mediated TNFR1 signal transduction prevents unwanted TNF responses in the central nervous system (CNS). ,, Highly selective small-molecule RIP1 inhibitors can penetrate the blood–brain barrier (BBB), indicating that these inhibitors are feasible targeted therapies for neuroinflammation and cell death. RIP1 has become a potential therapeutic target for the treatment of many human chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer’s disease (AD), and Parkinson’s disease (PD), as well as acute neurological diseases, such as cerebral ischemia, stroke, and traumatic brain injury. , At the intersection of cell death pathways and host-defense pathways, RIP1 may be leveraged for new therapeutic opportunities for immune and autoinflammatory diseases, such as inflammatory bowel disease (IBD), psoriasis, sepsis, rheumatoid arthritis (RA), and multiple sclerosis (MS), and cancers, including glioblastoma, melanoma, lung cancer, pancreatic cancer (PCa), gastric cancer, and gallbladder cancer …”

“…RIP1 has become a potential therapeutic target for the treatment of many human chronic neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), and Parkinson's disease (PD), as well as acute neurological diseases, such as cerebral ischemia, stroke, and traumatic brain injury. 8,9 At the intersection of cell death pathways and host-defense pathways, RIP1 may be leveraged for new therapeutic opportunities for immune and autoinflammatory diseases, such as inflammatory bowel disease (IBD), 10 psoriasis, 11 melanoma, lung cancer, pancreatic cancer (PCa), gastric cancer, and gallbladder cancer. 12 Drug design based on the catalytic and regulatory domains of the RIP1 kinase has been shown to be important to drug development.…”